4.2 Restriction enzymes and DNA ligases

Restriction enzymes and DNA ligases are essential tools in genetic engineering. These enzymes allow scientists to cut DNA at specific sites and join fragments together, enabling the creation of recombinant DNA molecules.

Understanding how these enzymes work is crucial for manipulating genetic material. Restriction enzymes create sticky or blunt ends, while DNA ligases join fragments by forming phosphodiester bonds. These techniques are fundamental for cloning, DNA analysis, and creating new genetic combinations.

Restriction Enzymes

Restriction Endonucleases and Recognition Sequences

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• Restriction endonucleases are bacterial enzymes that cleave DNA at specific recognition sequences
  • Protect bacteria from foreign DNA (bacteriophages)
  • Each restriction enzyme recognizes a specific 4-8 base pair palindromic sequence
• Recognition sequences are short, specific DNA sequences that restriction enzymes bind to and cut
  • Usually 4-8 base pairs long
  • Palindromic, meaning the sequence reads the same forward and backward on complementary strands (GAATTC)

Sticky and Blunt Ends

• Restriction enzymes can create sticky ends or blunt ends depending on their cleavage pattern
• Sticky ends have single-stranded overhangs that are complementary to other sticky ends
  • Allows for easy ligation of DNA fragments
  • Created by restriction enzymes that cleave asymmetrically (EcoRI)
• Blunt ends have no single-stranded overhangs and are flush
  • Harder to ligate than sticky ends
  • Created by restriction enzymes that cleave symmetrically at the center of the recognition sequence (SmaI)

DNA Ligation

DNA Ligase and Phosphodiester Bond Formation

• DNA ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent nucleotides
  • Joins DNA fragments together
  • Essential for recombinant DNA technology and molecular cloning
• Phosphodiester bonds are covalent bonds that link the phosphate group of one nucleotide to the sugar of the next
  • Form the backbone of the DNA double helix
  • DNA ligase catalyzes the formation of phosphodiester bonds between the 3' hydroxyl of one DNA fragment and the 5' phosphate of another

Applications of DNA Ligation

• DNA ligation is used to join DNA fragments together in molecular cloning
  • Allows for the creation of recombinant DNA molecules
  • DNA fragments with compatible ends (sticky or blunt) can be ligated together
• Ligation is also important in DNA repair processes
  • Joins Okazaki fragments during DNA replication
  • Repairs single-strand and double-strand breaks in DNA

DNA Analysis

Restriction Mapping

• Restriction mapping is a technique used to determine the positions of restriction sites within a DNA molecule
  • Helps to characterize and identify specific DNA sequences
  • Useful for planning cloning strategies and analyzing recombinant DNA
• Involves digesting DNA with one or more restriction enzymes and analyzing the resulting fragments
  • DNA fragments are separated by size using gel electrophoresis
  • The sizes of the fragments provide information about the positions of the restriction sites
• Restriction maps can be used to compare DNA sequences from different sources
  • Helpful in identifying mutations, polymorphisms, and evolutionary relationships
  • Can also be used to verify the identity and orientation of DNA inserts in recombinant plasmids